MOLECULAR MECHANISMS OF HIV 1 DNA INTEGRATION

所有逆转录病毒生命周期中的一个重要步骤，包括 人类免疫缺陷病毒(HIV)，是一种整合的双链病毒 将病毒基因组的DNA复制到宿主细胞的染色体中。二 整合过程中的关键因素是：病毒蛋白， 整合酶，以及线状病毒DNA末端的序列。整合酶 从线性病毒DNA分子的3‘端去除两个核苷酸， 并随后在过程中介导偶联的切割-连接反应 在目标DNA上进行交错切割，得到5‘ 靶DNA的末端共价连接到凹陷的3‘端 病毒DNA。病毒5‘端连接到靶点的时间 DNA目前还不清楚，涉及的蛋白质因素仍然是 被刻画出来。在整合过程中，宿主染色体中的许多位置 可以用作目标，尽管在集成方面有很大的差异 在目标站点中观察到了效率。这一机制 确定目标部位的专一性没有被很好地理解。自.以来 逆转录病毒复制需要整合，而且没有 在正常细胞功能中被认为是整合酶的对应物， 整合是开发特定抑制剂的一个有吸引力的目标 对抗逆转录病毒。这项提议的广泛、长期目标是 以加深对HIV整合机制的理解。这个 具体目标是(A)检查逆转录病毒过程中的靶点选择 DNA整合，以及(B)研究连接的动力学和机制 在病毒5‘端到靶DNA的最后一步中， 整合。实现这些目标的实验设计和方法 目标就是。(1)构建HIV-1与FIV的嵌合蛋白 用于确定选择DNA所涉及的蛋白质结构域的整合酶 用于整合的位点，并使用基于PCR的选择和扩增 一种确定整合酶最适DNA序列的方法 认识，(2)通过学习探索现场定向整合 体内外融合的活性和整合模式 由整合酶和序列特异性DNA结合组成的蛋白质 蛋白质，以及(3)使用一种新的策略来研究病毒3‘- 和5‘端连接，并建立了一种体外表征系统 5‘端连接步骤和所需的因素。这项研究可能会揭示 潜在的抑制剂新靶点并为药物提供有用的分析方法 放映。从研究整合选址中获得的信息 可能导致一种将外源基因插入特定基因的新方法 网站。

An essential step during the life cycle of all retroviruses, including human immunodeficiency virus (HIV), is integration of a double-stranded DNA copy of the viral genome into a chromosome of the host cell. Two factors are critical for the integration process: the viral protein, integrase, and sequences at the ends of the linear viral DNA. Integrase removes two nucleotides from the 3' ends of a linear viral DNA molecule, and subsequently mediates a coupled cleavage-ligation reaction during which a staggered cut is made in the target DNA, and the resulting 5' ends of the target DNA are covalently joined to the recessed 3' ends of the viral DNA. The timing of the joining of the viral 5' ends to target DNA is presently not known, and the protein factors involved remain to be characterized. During integration, many sites in the host chromosome can be used as targets, although a wide variation in integration efficiency is observed among the target sites. The mechanism that determines target site specificity is not well understood. Since integration is required for retroviral replication and there is no recognized counterpart to integrase in normal cellular function, integration is an appealing target for developing specific inhibitors against retroviruses. The broad, long-term objective of this proposal is to further the understanding of the mechanism of HIV integration. The specific aims are (A) to examine target site selection during retroviral DNA integration, and (B) to study the kinetics and mechanisms of joining of the viral 5' end to target DNA, a poorly characterized final step of integration. The experimental design and methods for achieving these goals arc. (1) to construct chimeric proteins between HIV-1 and FIV integrases for determining the protein domain involved in selecting DNA sites for integration, and to use a PCR-based selection and amplification protocol for determining the DNA sequence optimal for integrase recognition, (2) to explore site-directed integration by studying in vitro and in vivo the activities and integration patterns of fusion proteins consisting of integraSe and a sequence-specific DNA binding protein, and (3) to use a novel strategy to study the timing of viral 3'- and 5'-end joining, and to develop an in vitro system for characterizing the 5'-end joining step and the factors required. The study may reveal potential novel targets for inhibitors and provide useful assays for drug screening. Information obtained from studying integration site selection may lead to a new approach for inserting exogenous genes at specific sites.